As one example of conventional biosensors, a glucose sensor will be described below.
A generally known method of quantifying glucose is a system using a combination of glucose oxidase with an oxygen electrode or a hydrogen peroxide electrode. Glucose oxidase selectively oxidizes β-D-glucose, as a substrate, to D-glucono-δ-lactone, using oxygen as an electron mediator. During this reaction process, oxygen is reduced to hydrogen peroxide. Here, glucose is quantified either by measuring an amount of consumed oxygen with use of the oxygen electrode, or by measuring an amount of generated hydrogen peroxide with use of e.g. a platinum electrode.
However, such methods as described above are much influenced by dissolved oxygen concentration in the case of certain objects to be measured, or are impossible under conditions of oxygen being absent. Thus, a glucose sensor of such type has been developed that uses, as the electron mediator, a metal complex or an organic compound such as potassium ferricyanide, a ferrocene derivative and a quinone derivative without using oxygen as the electron mediator (See Japanese Laid-open Patent Publication Hei 2-062952).
For making this biosensor, an electrode system comprising a measurement electrode, a counter electrode, and a reference electrode is formed on an insulating base plate by e.g. screen printing, and an enzyme reaction layer including a hydrophilic polymer, an oxidoreductase, and an electron mediator is then formed on the electrode system. In case of need, a buffer is further added to this enzyme reaction layer.
When a sample solution containing the substrate is dropped onto the enzyme reaction layer of this biosensor, the enzyme reaction layer gets dissolved, and the enzyme reacts with the substrate, whereby this reaction causes the electron mediator to get reduced. The concentration of the substrate in the sample solution can be determined by an oxidation current for electrochemically oxidizing the thus reduced electron mediator after the end of the enzyme reaction.
According to this type of sensor, the reduced form of the electron mediator generated in consequence of the enzyme reaction is oxidized by the electrode, and the concentration of glucose can be determined by the oxidation current.
Such a biosensor can be used, in principle, for measuring various substances by using an enzyme whose substrate is each of the substances to be measured. For example, a serum cholesterol level, which is used as a diagnostic index in various medical institutions, can be measured by using, as oxidoreductase, cholesterol oxidase or cholesterol dehydrogenase and cholesterol esterase.
The enzyme reaction of cholesterol esterase progresses very slowly. By adding an appropriate surfactant thereto, cholesterol esterase can be enhanced in its activity, thereby to be able to shorten time needed for total reaction.
However, since this causes the reaction system to contain a surfactant, which badly affects hemocytes, it has been impossible to perform measurements using a whole blood.
As described above, in the case of measuring a cholesterol level in a blood, a surfactant is contained in the reaction system, and the surfactant badly affects erythrocytes in the blood. This has caused it impossible for sensors such as glucose sensors to measure a whole blood per se. A proposal has thus been made to provide a filter member in the vicinity of an opening of a sample solution supply pathway in order to supply thereto only plasma of a blood, with the erythrocytes having been filtered out. However, the speed of the flow of the filtrated plasma into inside of the sensor is low and inconstant, so that response values vary, and that bubbles are often generated when the plasma enters inside of the sensor, whereby measurement has been impossible.
An object of the present invention is to provide a so improved biosensor that does not have above described drawbacks, and allows plasma of a blood, with hemocytes thereof having been filtered out, to quickly reach the electrode system.
Another object of the present invention is to provide a cholesterol sensor, which is highly accurate and has superior response characteristics, and which allows a whole blood to be an object to be measured.